Effects of Peptone Supplementation in Different Culture Media on Growth, Metabolic Pathway and Productivity of CHO DG44 Cells; a New Insight into Amino Acid Profiles

Background: The optimization of bioprocess conditions towards improved growth profile and productivity yield is considered of great importance in biopharmaceutical manufacturing. Peptones as efficient sources of nutrients have been studied for their effect on media development; however, their role on metabolic pathway is not well understood. Methods: In the present study, the effect of different concentration of peptones on a recombinant Chinese hamster ovary (CHO) cell line grown in three serum-free suspension cultures was determined. Six peptones of different origins and available amino acid profiles were investigated regarding their impact on cell growth, productivity, and metabolic pathways changes. Results: In optimized feeding strategies, increases of 136% and 159% in volumetric productivity (for a low-nutrient culture media) and 55% (for a high-nutrient culture media) were achieved. Furthermore, particular sources of peptones with specific amino acid profile developed preferential results for each different culture medium. Two peptones, SoyA2SC and SoyE-110, were the only hydrolysates that showed production improvement in all three media. Casein Peptone plus Tryptone N1 and SoyA3SC showed different improved results based on their implemented concentration for each individual basal medium. Conclusion: The amino acid profile of peptones may provide clues to identify the most effective feeding strategies for recombinant CHO cells.


INTRODUCTION
ammalian expression systems are the most prevalently used hosts in recombinant protein production, due to their appropriate post-translational modifications. Nowadays, almost 60-70% of recombinant proteins are successfully produced in Chinese hamster ovary (CHO) cells mainly due to their correct post-translational modifications, and relatively easy adaptation to suspension growth in serum-free media for industrial scale bioreactors. The optimization of cell culture conditions towards improved growth and productivity of recombinant CHO (rCHO) cells is a critical step in biopharmaceutical process development and still faces a series of problems.
Commercial production of therapeutic proteins is now mainly based on serum-free media due to its economical advantages, beneficial effect on the downstream processing, and biological safety from infectious contaminants. Nevertheless, development of an optimized culture and medium condition has been a challenging debate and still no universal serum-free media applicable to all cell lines is available [1,2].
Recently, quite a large number of researches have been devoted to predictable media optimization with respect to its commercial outcomes for the biopharmaceutical industry.
Supplementation of hydrolysates has been shown to enhance cell growth and/or productivity in mammalian cells, such as rCHO cells [3,4], hybridoma [5], growth and enhance specific and volumetric productivity both in stable and transient expression systems [6,8,9] .
The mechanism of peptones' positive effects is not well understood. Previous studies have shown that they are substantial sources of nutrients and can provide cells with anti-apoptotic functions [10] as well as an stimulator in transfection efficiency of HEK293 cells [8].
However, the impact of peptone supplementation on metabolic behavior of rCHO cells is not properly known [11][12][13]. Furthermore, it is also unclear how each specific amino acid profile of a peptone is capable of altering metabolic attitude of the cells towards more glucose consumption or lactate and ammonia production. Therefore, the positive effect of peptones is still unpredictable and similar feeding strategies may cause different responses in various systems [14][15][16]. Consequently, a peptone-supplemented bioprocess needs to be optimized for a specific cell line or clone.
The ultimate aim at biopharma industry is to obtain optimized growth profile of cells along with higher yields of therapeutic protein production. Therefore, further understanding of intercellular changes during media supplementation is a promising approach for a predictable knowledge-based media development, which can lead to constancy in biopharma [17][18][19][20].
In the present study, a few plant-derived and caseinderived peptones ( Fig. 1 and Table 1) were investigated for their amino acid profile correlation with growth, productivity, biomass, and alterations in cell metabolic attitudes of a rCHO cell line. The two proprietary serum-free  media used were denominated CD DG44 and ProCHO  5 from Invitrogen (GIBCO Invitrogen, USA) and Lonza (Verviers, Belgium), respectively. The media were supplemented with 13.6 mg hypoxanthine l-1, 3.9 mg thymidine l-1, and 4 mM and 6 mM glutamine for ProCHO 5 and CD DG44 respectively. Furthermore, a basal medium based on RPMI 1640 (BRC-CD medium [BRC CDM]) was developed in the laboratory and supplemented with 44 mM glucose and 6 mM glutamine. All medium supplements used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chromolize t-PA (tissue plasminogen activator) Assay Kit was purchased from Biopool (Trinity Biotech PLC, Ireland). Packed cell volume (PCV) tubes and tube-spines were from (TPP, Techno Plastic Products AG, Trasadingen, Switzerland). Peptones were supplied from Organotechnie (La Courneuve, France) providing the total amino acid composition, molecular weight distribution, and free amino acid content of the peptones ( Fig. 1 and Table 1). Peptone stock solutions were prepared (20%, w/v), sterilized by filtration through 0.2 µm media filters, and stored at 4ºC.

Media and reagents.
Cell cultivation. The stable CHO DG44-derived cell lines, t-PA-producing cells from our previous studies [21][22][23]), were used in this study. Cultures were agitated at 110 rpm in TubeSpin® Bioreactors on an orbital shaker (at 37ºC in a % CO 2 atmosphere [24]. The cultures were inoculated with cells from the mid-   Cell number. Cell density and viability were assessed by the Trypan blue dye exclusion method using a hemocytometer (Neubauer improved, Brand). Cell viability was determined by the Trypan blue exclusion method (1:1 mixture of 0.2% trypan blue in a normal saline and cell sample). After cell counting, the remainder of each sample was centrifuged (at 5000 ×g for 1 min) to remove the cells, and the supernatant was frozen for further protein production and metabolite consumption/production rate analysis.

Biomass determination.
Biomass was determined by the PCV method using PCV tubes (TPP, Techno Plastic Products AG, Trasadingen, Switzerland) [25]. A cell density of 1 × 10 6 cells ml-1 was equivalent to a PCV of 0.25% for cells under standard cultivation conditions at 37°C.

Quantification (amidolytic activity test).
Biopool's Chromolize t-PA Assay Kit is a biofunctional immunosorbent assay based on capturing t-PA by sp-322 monoclonal antibodies coated on the microtest wells. After fulfilling, all the steps from the kit's manual samples were read at 405 nm and 492 nm.
Absorbance at 492 nm was measured and subtracted from 405 nm. Various dilutions of each sample were assayed. The amount of developed color is proportional to the amount of t-PA activity in the sample. Results are presented in amidolytic international that are defined as the amount of plasminogen activator required to release 1 pmol of pnitroanilide in 1 minute at 24°C at 405 nm in a I-cm path length, using an extinction coefficient of 9629 mol/L-' cm-' for p-nitroanilide.

Determination of CHO DG44 cell growth profile in different media.
To investigate media dependency of growth profile, the CHO clone was simultaneously cultivated in the same cultivation condition in three different chemically defined media, CD DG44, ProCHO 5, and our home-made BRC-CDM. Figure 2 show the growth profile of t-PA producing CHO DG44 cells based on cell count, viability, and PCV amounts. As shown in Figure 2, higher cell densities (Fig. 2a) and higher amounts of PCV ( Fig. 2b) were achieved in ProCHO 5 medium. Furthermore, cells tend to show almost similar growth in CD DG44 and BRC-CDM in terms of cell densities and biomass with slightly better results for CD DG44. Maximum cell density was on day 7 and culture duration was 11 days but after day 6, the viability of the cells was started to reduce (Fig. 2c). The same comparisons was made for CHO DG44 nontransfected cells in three media, and the growth profile was almost the same, except for slightly fewer maximum cell densities for non-transfected cells and the downward shift in viability, which for t-PA producing CHO DG44 takes place at day 7 and for non-transfected cells at day 9 (data not shown).  of 1 and 2 gl -1 . For ProCHO 5 medium, peptone supplementation resulted in a biomass growth in each of the supplemented peptones (Fig. 3). Regarding cell densities in both feeding concentrations, only peptone one in spite of a rise in PCV amounts led to reduced cell densities compared to control group. In 1 gl -1 feeding concentration, peptones 6, 5, 2, 4, and 3 showed an apparent rise in maximum cell densities up to 115, 59, 54, 45, and 20%, respectively ( Fig. 3a and   3b). This growth in cell numbers was even more drastic in 2 gl -1 feeding strategy; 156, 70, 65, 55, and 18% for peptones 6, 2, 5, 4, and 3, respectively ( Fig. 3c  and 3d). PCV values made significant rises with both peptone feeding strategies except for peptone 5, which led to reduced biomass in 2 gl -1 . In CD DG44 medium supplemented with both 1 and 2 gl -1 of peptones ( Fig.  4a and 4b), the positive impact of peptones on cell densities and biomass improvement was observed with peptones 1, 2, and 6. Maximum rise in cell densities was achieved with peptone 1 (43% increase for both feeding concentrations), followed by peptones 2 and 6.
Determination of the peptones effects on cell productivity. As mentioned above, the Chromolize t-PA assay ELISA based kit was used to determine the amount of total active form of t-PA protein. For quantitative determination of human t-PA activity in supernatant samples of transfected CHO cell, a biofunctional immunosorbent assay was performed. Based on Biopool's Chromolize t-PA assay kit, amidolytic unit was measured on day 9 of each culture. In ProCHO 5 medium with 2 gl -1 peptone concentration, peptones 5, 4, 6, and 3 showed a drastic rise in accumulated proteins on day 9 up to 55%, 51%, 41% and 35%, respectively. In 1 gl -1 , only peptone 1 represented an increased amount of 44% productivity on day 9, while the results for other peptones were either slight or even negative (Fig. 6a). On the other hand, in CD DG44 medium, 1 gl -1 concentration of peptones 4, 5, 6, and 2 could result in 136, 120, 104, and 22% increase in t-PA titers, while no significant improving results were seen in 2 gl -1 feeding concentration (Fig. 6b). As represented in Fig. 6c, for BRC-CDM, with 1 gl -1 concentration, peptones 6, 1, and 5 were capable to show increase of 139%, 60.36% and 33.64% for day 9 production yield respectively and the same as CD DG44, 2 gl -1 feeding concentration did not affect productivity.

Determination of metabolic behavior of t-PA producing CHO DG44 cells:
Metabolic changes in ProCHO 5 medium. Figure 7 illustrates the methabolic profile of rCHO cell lines in the presence of 2 gl -1 supplementation of peptones for ProCHO 5 medium. When utilized in 2 gl -1 concentrations, all six peptones maintained cells in higher amounts of glucose during culture durartion (Fig. 7a). In contrast, with 1 gl -1 concentration, peptones 1, 2, and 6 were able to supply cells with higher amounts of glucose during culture (data not shown). Regarding ammonia production (Fig. 7b), more fluctuating results were observed with different peptone supplements compared to control negative samples. The average ammonia accumulation on day 10 of culture was 0.51 and 0.85 gl -1 with 1 and 2 gl -1 peptone feeding concentrations, respectively. Lactate byproduct accumulation at the end of culture was greater with peptones 4, 5, and 6 ( Fig. 7c). Interestingly, a similar pattern of lactate production and final cumulative amounts were observed in two peptone supplement concentrations of 1 and 2 gl -1 ; 5.03 and 5.3 mM average final lactate concentrations, respectively.
Metabolic changes in CD DG44 medium. Figure 8 represents the metabolic profile changes in CD DG44 medium supplemented with 1 gl -1 of peptone concentration which was the selected feeding amount in terms of production titers in this media. Higher maintained glucose concentrations (Fig. 8a) were observed with all six peptones in both 1 and 2 gl -1 (data for 2 gl -1 not presented). In terms of ammonia production (Fig. 8b), peptone supplements resulted in higher ammonia concentartions up to day 6 with a shift to less ammonia amounts from day 6 to 10. The average ammonia accomulation on day 10 was 0.58 and 0.83 gl -1 with 1 and 2 gl -1 peptone feeding concentrations, respectively. Figure 8c shows the lactate production pattern during the culture. Except for peptone 3, all other five peptone supplements produced less lacatate amounts in 1 gl -1 . Final average lactate accomulation on day 10 were 4.18 and 5.46 mM in 1 and 2 gl -1 feeding concentrations.
http://IBJ.pasteur.ac.ir  Figure 9. This concentration was the selected feeding strategy, which led to better production amounts. No great effect on glucose consumption was observed with peptones feeding in this medium (Fig. 9a). However, in case of ammonia production (Fig. 9b), in spite of higher cumulative amounts with peptones 4 and 6, the average ammonia concentration on day 10 of culture with peptone feeding was reduced to 0.43 gl -1 compared to control non-fed culture (0.5 gl -1) .
No obvious change in lactate accomulation pattern with feeding peptones was observed in BRC-CDM (Fig. 9c). However, the final average amounts was slightly higher with peptone feeding compared to control samples (6.03 mM compared to 5.6 mM).

DISCUSSION
Peptones are widely used as supplements for serumfree culture media. The growth-promoting activity of peptones may have a dual effect in batch cultures, while it may promote rapid cell growth in the first days of culture. Also, it may lead to early depletion of vital nutrients and concomitant release of toxic metabolites, more rapid decline of culture viability, apoptosis and release of proteases which may degrade the product. In industrial processes, various strategies aiming to decrease cell growth during the production phase are routinely applied [28].
Similar studies on hydrolysate supplementation strategy have mainly focused on using statistical methods such as design of experiment (DoE) to determine the best mixture ratio to enhance productivity [29] regardless of peptones different amino acid profile. Thus, they have not considered the underlying story and need to be optimized for each protein and producing cell individually. However, the aim of the current study and the few similar papers [30] is to find a relationship between amino acid profile and production profile changes during media supplementation.
In a recent report, a proteomic approach was applied to identify CHO cells intracellular protein with either induced or suppressed expression upon peptones feeding [31]. The expression of several proteins involved in cell proliferation, metabolism, and protein folding/secretion was induced by peptone supplementation. However, other proteins involved in growth arrest and apoptosis induction were down-  regulated, suggesting that the growth-promoting effect of peptones acts through multiple molecular targets. These data supports that peptone impact on metabolic shift can also be traced in metabolic shifts of recombinant cells.
Although the molecular mechanisms of growth promoting effects of peptones are not fully understood, the positive effects observed could be considered as a consequence of the diverse amino acid composition of the peptones [32][33][34][35]. However, the relation between their amino acid content and their role in growth, production, and metabolic behavior of recombinant cells are poorly understood.
Here, we propose that peptone supplementation effects are correlated to amino acid profile, which resulted in metabolic behavior alterations during cultures supplemented with peptones. The peptones used in this study were intentionally chosen among commercially available peptones of plant and casein origin with available total and free amino acid content and molecular distribution on the ground that a correlation exists between amino acid profile and the resulted cell attitude. This approach would be a good start point for further detailed analysis, determination and as a final goal prediction of the best strategy for media optimization. The effect of peptone-mediated improvement on a basal serum-free medium was analyzed by assessing the growth, productivity, and metabolic behavior of a rCHO clone.
The growth and productivity promoting effect of peptones is dependent on the basal medium nutrient composition. The results of cultivation in three different basal media (ProCHO 5, CD DG44, and BRC  CD) showed that t-PA producing CHO cell line represents privilege for ProCHO 5 compared to CD DG44 and BRC-CDM in terms of maximum achieved cell densities and biomass amount (Fig. 2). This data is convincing enough because ProCHO 5 as a highnutrient medium is optimized for recombinant protein production in suspension adapted cells. On the other hand, CD DG44 and BRC CD (our house-made) as low-nutrient media show a similar growth profile, which is significantly lower in terms of cell densities and biomass amounts. Peptone supplementation strategies are supposed to show more distinguished effects in a low-nutrient medium compared to a highnutrient one.
Determining the optimum peptone feeding concentration is a complicated step towards media supplementation. Our data suggests that a specific peptone concentration should be set for each basal  medium. As shown in Figures 3, 4, 5, and 6, for a highnutrient medium such as ProCHO 5, the higher concentration of peptones (2 gl -1 ) tend to have higher production-promoting effects. Also regarding cell densities, 2 gl -1 is the concentration of choice in this medium with higher cell densities achieved in comparison to 1 gl -1 . In contrast, for low-nutrient media, such as CD DG44 and BRC CD, 1 gl -1 concentration leads to sharper effects. Concerning protein production titers, both media showed distinctly improved results only in 1 gl -1 .
These results also confirm that while focusing on production titers, a definite set of peptones shows a privilege in each basal medium, which is totally different in terms of prioritization. In other words, in ProCHO 5, peptones 5, 4, 6, and 3 are selected and prioritized in the mentioned order, while in CD DG4, the order goes for 4, 6, 5, and 2 and in BRC CD is 5, 1, 6, and 2. The story behind these differences is the key factor for predicted media optimization.
In the current study, we observed that peptones from the same source casein/soy do not necessarily produce similar results for a specific media. In ProCHO 5 medium for example, two casein-based peptones (peptones 3 and 4) and two soy-based peptones (peptones 5 and 6) showed an apparent increase in productivity. In CD DG44, all the three soy peptones (6, 5, and 2) were preferred but one of the cases in peptones (4) was able to result in higher production titers. BRC-CDM is preferably enriched with soy peptones (6 and 5) and one of the cases in origins (peptone1). This led us to have a closer look at amino acid profile of each of the six peptones.
Peptone 1 which shows effect in BRC CD is different from other casein peptones in its aspartic acid content (Fig. 1), which is more close to soy peptone origins rather than casein ones. The selected casein peptone for CD DG44, peptone 4, is different from other casein peptones regarding its higher methionine amounts.
It is worth mentioning that in ProCHO 5 medium when peptones are utilized in a low concentration (1 gl -1 ), only peptone 1 with high aspartic acid can result in improving production yields. This peptone shows its effect with biomass increase in spite of reduced cell numbers. It also provides the cells with improved metabolic profile, reduced glucose consumption, and ammonia and lactate production (data not shown). On the other hand, peptone 6 in this concentration (1 gl -1 ) reaches much higher cell densities with a sharp reduction in glucose consumption. Nevertheless, it is not able to give a drastic rise to production titers, which may be due to high amounts of lactate produced (data not shown). Furthermore, it was observed that 2 gl -1 peptones as a source of nitrogen, produce average amounts of 0.85 gl -1 of ammonia byproduct on day 10, which is above the inhibitory range (0.6 gl -1 ) [36]. However, these supplements are still capable of production improvements in this concentration (Figs. 6 and 7).
Regarding CD DG44 medium with 1 gl -1 , all peptones except for peptone 3 reduce glucose consumption rate during culture. Moreover, this is the only peptone that produces a high amount of lactate byproduct in comparison to the other peptones. Peptone 3 is different from other casein-based peptones in which it lacks cysteine, methionine, and tryptophan. This peptone also contains some amounts of glutamine, anserine, and citrulline. These differences are possibly the reason for its various effects on metabolic pathway and its negative impact on protein production in this media.
In BRC CD besides two soy peptones (5 and 6), the "casein peptone plus" with highly different aspartic acid amount affects cells productivity in a positive manner. This medium as the less nutrient basal medium results in the maximum production increase effect of 159%, while in CD DG44 cells, the greatest titer rise is 136%. Except for higher accumulation of ammonia, no drastic change in metabolic attitude was observed with peptone supplementation in this special medium. It is also interesting to stress that peptone supplementation is not the reason for reduced cell proliferation or productivity since the accumulation of ammonia and lactate is less than the levels reported in the literature as being inhibitory (18 mM of lactate [37] and 8 mM of ammonia [36,38]). Based on previous studies, positive effect of peptones on the production pattern in t-PA producing CHO cells may be attributed to scavenging of proteolytic activities produced by the cells from the peptone peptides [39].
In this study, it was observed that peptone 6, a soybased peptone with a higher amount of glutamic acid compared to other soy peptones (Fig. 1), and also composed of some amounts of glutamine, asparagines, and citrulline, is the only peptone improving productivity with all media and in every two concentrations used. This data corroborates previous investigations on improving effect of glutamine in t-PA production [39,40].
High ammonia concentration (up to 7.5 mM) has been shown to have negative impact on t-PA production [38,41]. However, in the cumulative concentrations achieved by peptones in this study, the ammonia inhibition does not seem to be a key factor for this cell line as seen with many others [39].
As shown in Table 1, the total amino acid contents of the peptones studied did not exceed about 80% in mass, with soy-derived peptones containing the lowest percentage of amino acids (about 50%). We can therefore suppose that the observed effects may be due at least in part to other nutrients than amino acids, which are possibly present in the hydrolysates. Previous studies also revealed that peptone hydrolysates may contain a variable proportion of other nutrients, such as sugars, lipids, vitamins, nucleic acids, and minerals [42][43][44][45].
All peptones tested in the present study had a similar molecular weight distribution, with over 80% of all fractions below 1 kDa (Table 1) and almost 100% below 10 kDa. Lower molecular weight fractions of plant peptones have been proven to better support cell growth and prolong cell viability, besides facilitating downstream product recovery [46].
The cultivation of rCHO cells in peptonesupplemented, serum-free media is a widely established cultivation method for the production of therapeutic proteins. The data presented in this study suggest that based on amino acid profile of peptones and its compatibility with basal media, cell-line tailored feeding strategy can be developed to improve the productivity of rCHO cell lines in basal media.